Effects of vimentin clustering on the keratin-8 network across cell lines. (A, C, and E) Indicated cell lines were co-transfected with Vim-mCh-FKBP and FRB-BFP-GCN4-ppKin14 constructs (transfected cells are outlined), while non-transfected cells served as controls. After 1 h of treatment with or without rapalog, the cells were fixed for analysis. Total vimentin and keratin-8 network intensities were measured after staining with anti-vimentin and anti–keratin-8 antibodies, respectively. The white dashed box indicates the region of the cell enlarged in zoom images. Images were obtained with Airyscan microscopy. (B, D, and F) Colocalization analysis in COS-7 (B), U2OS (D), and HeLa (F) cells. The graphs represent Manders’ coefficients of thresholded images, measured from 50.2 × 50.2-µm ROIs per COS-7 cell (B), 45.11 × 45.11-µm ROIs per U2OS cell (D), and 35.03 × 35.03-µm ROIs per HeLa cell (F). Graphs show mean ± SD, with individual cell measurements represented by dots. In B, data were collected from n = 25–29 cells; in D, n = 27–29 cells; and in F, n = 27–34 cells across three independent experiments. ns, not significant; ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗∗, P < 0.0001 based on Kruskal–Wallis statistical analysis.